Phylogenetic analysis of the Trypanosoma genus based on the heat-shock protein 70 gene.

Trypanosome evolution was so far essentially studied on the basis of phylogenetic analyses of small subunit ribosomal RNA (SSU-rRNA) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes. We used for the first time the 70kDa heat-shock protein gene (hsp70) to investigate the phylogenetic relationships among 11 Trypanosoma species on the basis of 1380 nucleotides from 76 sequences corresponding to 65 strains. We also constructed a phylogeny based on combined datasets of SSU-rDNA, gGAPDH and hsp70 sequences. The obtained clusters can be correlated with the sections and subgenus classifications of mammal-infecting trypanosomes except for Trypanosoma theileri and Trypanosoma rangeli. Our analysis supports the classification of Trypanosoma species into clades rather than in sections and subgenera, some of which being polyphyletic. Nine clades were recognized: Trypanosoma carassi, Trypanosoma congolense, Trypanosoma cruzi, Trypanosoma grayi, Trypanosoma lewisi, T. rangeli, T. theileri, Trypanosoma vivax and Trypanozoon. These results are consistent with existing knowledge of the genus' phylogeny. Within the T. cruzi clade, three groups of T. cruzi discrete typing units could be clearly distinguished, corresponding to TcI, TcIII, and TcII+V+VI, while support for TcIV was lacking. Phylogenetic analyses based on hsp70 demonstrated that this molecular marker can be applied for discriminating most of the Trypanosoma species and clades.

Chemistry, cytotoxicity and antileishmanial activity of the essential oil from Piper auritum.

Leishmaniasis is one of the most important parasitic infections, but current treatments are unsatisfactory due to their toxicity, cost and resistance. Therefore, the development of new antileishmanial compounds is imperative. Many people who live in endemic areas use plants as an alternative to treat the disease. In this paper, we characterised the essential oil from Piper auritum, evaluated its cytotoxicity and determined its antileishmanial activity. The chromatogram obtained by gas chromatography revealed 60 peaks and we found that safrole was the most abundant compound, composing 87% of the oil. The oil was active against the promastigotes of Leishmania major, Leishmania mexicana, Leishmania braziliensis and Leishmania donovani with a favourable selectivity index against peritoneal macrophages from BALB/c mice. The Piper-oil inhibited the growing of intracellular amastigotes of L. donovani with an IC50 value of 22.3 +/- 1.8 microg/mL. This study demonstrates the usefulness of the essential oils as a promising alternative to treat leishmaniasis.

Chemistry, cytotoxicity and antileishmanial activity of the essential oil from Piper auritum

Leishmaniasis is one of the most important parasitic infections, but current treatments are unsatisfactory due to their toxicity, cost and resistance. Therefore, the development of new antileishmanial compounds is imperative. Many people who live in endemic areas use plants as an alternative to treat the disease. In this paper, we characterised the essential oil from Piper auritum, evaluated its cytotoxicity and determined its antileishmanial activity. The chromatogram obtained by gas chromatography revealed 60 peaks and we found that safrole was the most abundant compound, composing 87 percent of the oil. The oil was active against the promastigotes of Leishmania major, Leishmania mexicana, Leishmania braziliensis and Leishmania donovani with a favourable selectivity index against peritoneal macrophages from BALB/c mice. The Piper-oil inhibited the growing of intracellular amastigotes of L. donovani with an IC50 value of 22.3 ± 1.8 ìg/mL. This study demonstrates the usefulness of the essential oils as a promising alternative to treat leishmaniasis.

Effect of oral treatment with the essential oil from Chenopodium ambrosioides against cutaneous leishmaniasis in BALB/c mice, caused by Leishmania amazonensis.

BACKGROUND: The aromatic herb Chenopodium ambrosioides is widely known for its antiparasitic activity. The aim of this study was to investigate the antileishmanial effect of Chenopodium oil administered by the oral route at different doses and to compare its action to conventional, clinically used drugs. MATERIALS AND METHODS: BALB/c mice were infected with Leishmania amazonensis and treated with 30, 60, 90, 120, and 150 mg/kg of the essential oil for 15 days. A second experiment was performed to compare the antileishmanial effect of Chenopodium oil with glucantime (28 mg/kg), amphotericin B (1 mg/kg), and pentamidine (4 mg/kg), which were administered daily over 15 days by the intraperitoneal route. RESULTS: Statistically significant differences were observed between BALB/c mice treated with all the doses of the product compared with untreated animals and the mice treated with the vehicle. A dose of 150 mg/kg was the most effective and no macroscopic toxic effects were observed. The size of lesion showed a linear correlation at each point (R > 0.8322), with a 50% effective concentration of 51.4 mg/kg. At 150 mg/kg, the essential oil showed better activity compared with animals treated with glucantime, amphotericin B, and pentamidine. CONCLUSION: C. ambrosioides caused a promising therapeutic effect against cutaneous leishmaniasis caused by L. amazonensis, which could be explored to develop a new alternative treatment for cutaneous leishmaniasis.

PCR-RFLP y RAPD para la tipificación de Leishmania neotropical / PCR-RFLP and RAPD for typing neotropical Leishmania

Introducción. El análisis de la longitud de los fragmentos de restricción del producto amplificado y el estudio del ADN polimórfico amplificado al azar han demostrado ser herramientas útiles para la tipificación de Leishmania. Objetivos. Estudiar la utilidad de las técnicas moleculares para la identificación y tipificación de cepas de referencia de Leishmania spp. del Nuevo Mundo y valorar su aplicabilidad a muestras clínicas. Materiales y métodos. Se aplicó PCR para amplificar el gen que codifica la cisteíno-proteinasa B, y el análisis de la longitud de los fragmentos de restricción del producto amplificado utilizando ácido desoxirribonucleico de 16 cepas de referencia de Latinoamérica y de muestras clínicas de pacientes colombianos con leishmaniasis, y la técnica del ácido desoxirribonucleico polimórfico amplificado al azar utilizando ocho cepas de referencia. Se establecieron los patrones de bandas en cada caso. Resultados. Se obtuvo producto de amplificación en la PCR para Leishmania braziliensis, L. peruviana, L. panamensis y L. guyanensis. Para el resto, no fue posible amplificar el gen con los cebadores utilizados. La restricción mostró un patrón de bandas común para L. peruviana, L. guyanensis y L. panamensis, mientras L. braziliensis, presentaba un perfil individual único. El análisis de restricción del producto amplificado generó un patrón de bandas similar en los cinco pacientes estudiados, que se correspondía con el patrón generado por L. peruviana, L. guyanensis o L. panamensis. Mediante la amplificación al azar se obtuvieron patrones de bandas reproducibles con todas las cepas estudiadas, que posibilitaron la diferenciación. Se discuten las ventajas y limitaciones de ambos procederes. Conclusiones. El combinar ambas metodologías resultaría útil para identificar especies de importancia médica, tomando en cuenta sus ventajas y desventajas.

[PCR-RFLP and RAPD for typing neotropical Leishmania]. / PCR-RFLP y RAPD para la tipificación de Leishmania neotropical.

INTRODUCTION: The analysis of the PCR-restriction fragment length polymorphism and random amplified polymorphic DNA have been useful tools for Leishmania identification. OBJECTIVES: Molecular procedures were demonstrated for identification and typing of reference strains of New World Leishmania and their applicability was validated for clinical samples. MATERIALS AND METHODS: DNA was extracted from 16 reference strains of Latin American Leishmania as well as from clinical samples of leishmaniasis patients. A sequence coding for cysteine proteinase B was amplified by PCR and subjected to restriction fragment length polymorphism analysis. The enzyme used was Taq1. For eight of the reference strains, the random amplified polymorphic desoxyribonucleic acid technique (RAPD) was applied. Band patterns for Leishmania species differentiation were established each each method. The sample size of the clinical sample was of 5. RESULTS: PCR products of the cysteine proteinase B gene were obtained for L. braziliensis, L. peruviana, L. panamensis and L. guyanensis. For the other species, L. mexicana, L. amazonensis, L. garnhami, L. lainsoni, L. chagasi, L. naiffi, no amplification occurred. The patterns of restriction fragments revealed band patterns in common for L. peruviana, L. guyanensis and L. panamensis, whereas L. braziliensis had a distinctive pattern. When human samples were examined, amplification occurred for all cases, and the profiles corresponded to the common profile of L. peruviana, L. guyanensis and L. panamensis. The RAPD technique demonstrated reproducible and distinctive patterns for each of the 8 reference strains, L. mexicana, L. amazonensis, L. garnhami, L. lainsoni, L. chagasi, L. naiffi, making possible to differentiate all them. The advantages and limitations of each procedure are discussed. CONCLUSIONS: The combination of RFP and RAPD methodologies provide useful tools to identify medical important species of Leishmania by recognizing DNA sequences characteristic of each species.

Combined effect of the essential oil from Chenopodium ambrosioides and antileishmanial drugs on promastigotes of Leishmania amazonensis.

To date, there are no vaccines against Leishmania, and chemotherapy remains the mainstay for the control of leishmaniasis. The drugs of choice used for leishmaniasis therapy are significantly toxic, expensive and with a growing frequency of refractory infections. Because of these limitations, a combination therapy is the better hope. This work demonstrates that the essential oil from Chenopodium ambrosioides shows a synergic activity after incubation in conjunction with pentamidine against promastigotes of Leishmania amazonensis. However, an indifferent effect has been found for combinations of meglumine antimoniate or amphotericin B and the essential oil.

Combined effect of the essential oil from Chenopodium ambrosioides and antileishmanial drugs on promastigotes of Leishmania amazonensis

To date, there are no vaccines against Leishmania, and chemotherapy remains the mainstay for the control of leishmaniasis. The drugs of choice used for leishmaniasis therapy are significantly toxic, expensive and with a growing frequency of refractory infections. Because of these limitations, a combination therapy is the better hope. This work demonstrates that the essential oil from Chenopodium ambrosioides shows a synergic activity after incubation in conjunction with pentamidine against promastigotes of Leishmania amazonensis. However, an indifferent effect has been found for combinations of meglumine antimoniate or amphotericin B and the essential oil.

Até hoje não temos vacina contra a Leishmania e a quimioterapia é a indicação para o controle desta doença. Os remédios que hoje utilizamos são tóxicos e muito caros e além disso o resultado não é sempre o desejado. Por isso, uma terapia de combinação é a melhor opção. Este trabalho mostra que o óleo de essência de C. ambrosioides tem atividade sinérgica junto com a pentamidina sobre os promastigotas de L. amazonensis, diferente do resultado da combinação de antimônio de meglumine e anfotericina B e o óleo de essência.

PCR-RFLP/Hsp70 para identificar y tipificar Leishmania de la región neotropical / PCR-RFLP/Hsp70 for identification and tipification of Leishmania from the tropical region

Se realizó la estandarización de las condiciones de amplificación del gen que codifica para la proteína de choque térmico de 70 kDa (Hsp70) de Leishmania mediante la reacción en cadena de la polimerasa (PCR-Hsp70), así como el análisis posterior de la longitud de los fragmentos de restricción (RFLP) del producto amplificado, utilizando como molde ADN puro de una cepa de referencia de Leishmania mexicana. Se estudió la sensibilidad y especificidad analíticas de la PCR, así como la reproducibilidad, utilizando ADN de L. mexicana, L. amazonensis, L. guyanensis y L. lainsoni. Se obtuvo una banda de 1,3 Kpb, demostrándose la amplificación del gen que codifica para la Hsp70. Los patrones de bandas obtenidos tras la digestión enzimática, utilizando la enzima Hae III, permitieron establecer diferencias entre las especies estudiadas: L. guyanensis y L. lainsoni se diferencian entre sí y estas a su vez de L. mexicana y L. amazonensis, que mostraron un patrón de bandas común. La sensibilidad y especificidad analíticas de la técnica fueron adecuadas. Se demostró la factibilidad de identificar y tipificar especies del continente americano mediante la PCR-RFLP/Hsp70, y de utilizar la restricción enzimática del producto amplificado para distinguir entre Leishmania spp. y Trypanosoma cruzi, dándose un primer paso en el establecimiento de estos métodos moleculares en el laboratorio de referencia del instituto.

Necesidad del uso de cromógenos para cuantificar promastigotes de Leishmania en placas de 96 pozos / The need for the use of chromogens for quantifying Leishmania promastigotes on plates from 96 wells

The existing difficulties in the treatment of leishmaniasis justify the testing of the effect of new products on parasite forms in the search of a therapeutic alternative for the parasitosis. Given the need of establishing a method to evaluate the activity of natural synthetic products in vitro under the Cuban conditions, this paper was aimed at defining the usefulness of p-nitrophenilphosphate as a chromogenic substance for quantification of parasites in plaques from 96 wells. To standardize this colorimetric method the stages of the parasite growth curve were set. The study of linearity and selection of the sample size, which was optional for these assays, showed that it was possible to obtain maximum linear determination coefficient with 20 mm. Likewise, the variation coefficient was compared with and without the chromogen and the effect of changes in culture medium on the reading of absorbances was analyzed. The set limit of quantification proved the need of using chromogen for the purposes of this paper and the general results allow to recommend this less subjective, simpler and quicker methodology to test products of interest in this field.

Enzymeba, Procedimiento eficaz para estudiar la prevalencia de infección intestinal por entamoeba histolytica / ENZYMEBA: an effective method for analzing the prevalence of Entamoeba histolytica intestinal infection

Por la unidad epidemiológica que tiene el desarrollo de procedimientos que permitan estudios confiables de prevalencia de amebiosis intestinal se estudió la eficacia de ENZYMEBA, ensayo inmunoenzimático para la detección en heces de Entamoeba histolytica. ENZYMEBA, mediante el empleo de una sola muestra por individuo participante en el estudio, demostró infección por E. histolytica en 27 de los 686 casos estudiados. La observación microscópica de heces sólo igualó los resultados de ENZYMEBA cuando fueron analizados los resultados de ambos procedimientos sobre 3 muestras de cada uno de los individuos en el estudio. Por su alta sensibilidad, requerir de una sola muestra por persona encuestada y ser un inmunoensayo realizable en condiciones mínimas de laboratorio, consideramos que ENZYMEBA es una herramienta muy útil para estudios de prevalencia de amebiosis intestinal